IN BRIEF: To save endangered rhinoceros species, assisted reproductive technologies are warranted. We here report in vitro blastocyst generation of the Near-Threatened Southern white rhinoceros and, for the first time, also of the technically Extinct Northern white rhinoceros. ABSTRACT: The Anthropocene is marked by a dramatic biodiversity decline, particularly affecting the family Rhinocerotidae. Three of five extant species are listed as Critically Endangered (Sumatran, Javan, black rhinoceros), one as Vulnerable (Indian rhinoceros), and only one white rhino (WR) subspecies, the Southern white rhinoceros (SWR), after more than a century of successful protection is currently classified as Near Threatened by the IUCN, while numbers again are declining. Conversely, in 2008, the SWR's northern counterpart and second WR subspecies, the Northern white rhinoceros (NWR), was declared extinct in the wild. Safeguarding these vanishing keystone species urgently requires new reproductive strategies. We here assess one such strategy, the novel in vitro fertilization program in SWR and - for the first-time NWR - regarding health effects, donor-related, and procedural factors. Over the past 8 years, we performed 65 procedures in 22 white rhinoceros females (20 SWR and 2 NWR) comprising hormonal ovarian stimulation, ovum pick-up (OPU), in vitro oocyte maturation, fertilization, embryo culture, and blastocyst cryopreservation, at an efficiency of 1.0 ± 1.3 blastocysts per OPU, generating 22 NWR, 19 SWR and 10 SWR/NWR hybrid blastocysts for the future generation of live offspring.

AVANTEA Laboratorio di Tecnologie della Riproduzione Lombardy Cremona Italy

Department of Comparative Biomedicine and Food Science Università degli Studi di Padova Italy

Department of Stem Cell Biology and Medicine Kyushu University Maidashi Higashiku Fukuoka Japan

Fondazione Avantea Riproduzione Cremona Lombardy Cremona Italy

Freie Universitat Berlin Veterinary Medicine Berlin Germany

Graduate School of Medicine Osaka University Suita Osaka Japan

Kenya Wildlife Service Veterinary and Capture Services Nairobi Kenya

Leibniz Institute for Zoo and Wildlife Research in the Forschungsverbund Berlin eV Reproduction Management Alfred Kowalke Straße Berlin Germany

Max Delbrück Center for Molecular Medicine in the Helmholtz Association Technology Platform Pluripotent Stem Cells Berlin Germany

Ol Pejeta Conservancy Conservation Laikipia Nanyuki Kenya

Universita degli Studi di Padova Ethics Laboratory for Veterinary Medicine Conservation and Animal Welfare Veneto Padova Italy

Wildlife Training and Research Institute Nakuru County Naivasha Kenya

ZOO Dvůr Králové Communication and International Projects Štefánikova Dvůr Králové nad Labem Czech Republic

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